Medium-term storage of calf beddings affects bacterial community and effectiveness to inactivate zoonotic bacteria

Land-spreading of animal faecal wastes -such as animal beddings- can introduce zoonotic enteropathogens into the food system environment. The study evaluated the effectiveness of animal beddings naturally contaminated by calf manure to reduce E. coli O157:H7 or Salmonella enterica. The two pathogens were introduced separately as a four strains-cocktail and at high (>6.5 Log10 g-1) concentration into bedding materials, and their inactivation over a 10 weeks-period was monitored by using a Most Probable Number (MPN) enumeration method. Inactivation of E. coli O157:H7 was more effective in the bedding inoculated immediately after collection from calf pens than in the beddings inoculated after a 2 months-pre-storage period: E. coli O157:H7 levels were reduced by 6.6 Log10 g-1 in unstored bedding (0.5 Log10 g-1 recovered; 95%CI: 0.0–1.2), and by 4.9 Log10 g-1 in pre-stored bedding (2.2 Log10 g-1 recovered; 95%CI: 1.5–2.8) with a significant (p<0.05) difference between unstored and pre-stored. S. enterica was inactivated less effectively as counts were reduced by one order of magnitude, with no significant difference in inactivation between unstored and pre-stored beddings. Low levels of naturally occurring E. coli O157 and Salmonella spp. were detected in the non-inoculated beddings, as well as in the straw prior to use in the animal facility. To better understand the possible biological processes involved, the bacterial community present in the beddings was characterised by short-read 16S rRNA sequencing. Pre-storage of the bedding affected the composition but not the diversity of the bacterial community. Analyses of the key bacterial phyla suggested that the presence of a diverse and stable bacterial community might facilitate inactivation of the introduced pathogens, and a possible role of bacterial orders associated with lignocellulolytic resources. Overall, the study contributed to the understanding of the fate of zoonotic bacteria introduced in animal beddings during storage and identified bedding storage practices pre-and post-use in animal facilities that could be important to prevent the risk of zoonosis dissemination to the environment or to the dairy herds.


Introduction
World dairy and beef production has undergone significant growth in the last 20 years and is projected to keep growing globally over the next decade, due to high population growth in some areas and a desire for high-protein food [1].This demand will likely be met by intensification of farm practices and increased reliance on housing systems with bedding materials on which the animals walk, stand, lie, defecate and urinate.In dairy housing systems, natural materials such as straw, woodchips, wood shavings or sawdust are commonly used as organic bedding materials [2,3].These materials ensure cow comfort and can also be recycled as agricultural soil amendments providing nutrients to plants and soils, reducing the need for synthetic fertilizers, and contributing positively to soil quality and crop health [3][4][5].However, animal manure and used beddings soiled with animal feces and urine can also be a potential reservoir for enteric pathogens and, if not handled appropriately, pose a significant risk to animal and human health [6,7].Among the enteric pathogens commonly detected in used animal beddings are Escherichia coli O157:H7 and Salmonella spp.[8,9].These bacteria are leading causes of foodborne illness outbreaks associated with consumption of meat, milk or fresh produces contaminated with animal manure [10,11].They have also been identified in outbreaks associated with amendments of vegetable fields with used animal beddings, or with direct contact of humans with used animal beddings [12][13][14].
Alongside control of these pathogens in the animals themselves, pre-treatment of cattle waste -including used bedding materials-prior to recycling on-farm has been promoted to reduce the spread of enteric pathogens [15].The liquid fraction of animal wastes can be separated from the solid fraction, which itself can be used immediately or further maturated through different methods, including stock-piling or composting.Stockpiling of soiled animal beddings, also known as "static piling" or "passive composting", consists of heaping or stacking used bedding or separated solids, either inside or outside animal facilities [16].It is a simple storage practice used routinely by many farmers [17].Compositing, which requires actively incorporating dry matter, managing moisture content, and regular mixing/aerating in order to mature animal wastes into high-value soil amendments, has the disadvantage to be challenging technically and costly for farmers [17,18].Adequate pre-treatment of used beddings is critical for the farm sustainability, as unproper pre-treatment of used beddings before re-use or land spreading has been shown to increase the prevalence of zoonotic micro-organisms in dairy herds [19].Maturation and recycling of used animal beddings raised questions around the effect of recontamination through addition of freshly soiled bedding to the storage heap.Stored manure undergoes degradation during their maturation, with accompanying variations in chemical and/or structural composition as well as in the predominant microorganisms [20,21], and there is currently no information on the inactivation of zoonotic bacteria if re-introduced in the beddings during the storage and maturation period.
Inactivation of E. coli O157:H7 or Salmonella spp.during the storage of stockpiles or maturation of composts was reported to vary substantially among studies, ranging from weeks to months [22,23].The effects of the heaps physical and chemical characteristics and the atmospheric conditions on pathogen inactivation has received considerable attention, as reviewed by Ongeng et al [24]].There are comparatively few reports and limited understanding of the biological mechanisms that account for inactivation of foodborne pathogens.For example, You et al. [25] indicated that indigenous but unidentified bacteria in manure can exert an antagonist effect on the persistence of pathogens.Franz et al. [26] showed that the overall survival time of E. coli O157:H7 in cattle manure was negatively correlated with the number of coliforms.Semenov et al. [27], who characterized the bacterial community in manure by DGGE analyses, could not establish a strong relationship between bacterial community composition and survival time of E. coli O157:H7.
The aims of this study were (1) to compare the inactivation of E. coli O157:H7 and S. enterica when introduced in animal beddings before or during storage; and (2) to better understand the possible biological processes involved in the inactivation of E. coli O157:H7 and S. enterica by characterizing the bacterial community present in the bedding.The bedding material was sourced from a calf rearing facility and consisted of straw naturally contaminated by dairy calves' excreta (e.g., feces, urine, hair, saliva, etc.).Straw without excreta was used to investigate the effect of the bacterial community naturally present in the straw.We hypothesized that both the inactivation of the food-borne bacteria and the diversity and composition of the bacterial community would be different between the straw-based materials and with or without prestorage.

Bacterial strains
Eight strains (four E. coli O157:H7 and four Salmonella enterica subsp.Enterica) isolated from dairy animals (calf feces and dairy effluents) and their environment (calf bedding or bird droppings), and fully characterized by whole genome sequencing (BioProject PRJNA933223) were used for the study.Each strain was revived from cryobeads (Microbank TM , Ngaio Diagnostics, Nelson, New Zealand) using Sheep blood agar (Fort Richard Laboratories, Auckland, New Zealand) and Tryptic Soy Agar (TSA; Fort Richard Laboratories) at 35˚C as previously described [28].All the bacterial growth collected from TSA was resuspended into 50 ml of 0.85% saline.Bacterial cocktails for inoculation of bedding material were freshly prepared by combining each undiluted bacterial suspensions in equal proportions, resulting in a 120 mL of S. enterica cocktail and a 120 mL of E. coli O157:H7 cocktail.The concentration of each pure culture was confirmed by serial dilution in 0.1% peptone and enumeration by plate counts on Sheep Blood agar after 24-h incubation at 35˚C.

Bedding collection and processing
Two straw-based beddings were used for the experiment.The "straw + manure" bedding was sourced from one pen of the fully covered calf rearing facility of Dairy Farm 4 at Massey University (Palmerston North, New Zealand).Sampling did not require a field permit consent but complied with the farm's health and safety requirements and biosecurity policies.The straw was collected by bulking bedding samples (200-400g each) from 40 locations in the pen, avoiding water and feed troughs, and from the top (5 cm) and bottom (25 cm) layers of the bedding.The pen from which bedding was collected housed 40 unweaned (<2-month-old) calves at collection time but had been used for the entire duration of the calving season (approximately 6 weeks; 100 calves in total).The "straw + manure" bedding contained straw, fresh calf urine and fecal material and had a low level of visible body material (such as calf hair).The "straw" was collected from the middle (one location) of an unopened bale stored in another covered shed at the same farm.Care was taken to avoid the outside layer, which was unprotected from the elements and from the birds.Both bedding materials were collected using sterile gloves.The collected bedding samples (~2 kg (wet weight) each) were immediately transported in insulated containers to the laboratory.The water content of the collected bedding sample estimated by gravimetry was 63% and 9% for the straw + manure and the straw, respectively.Each bedding sample was cut into 2-5 cm long pieces using sterile scissors and mixed thoroughly using the quarterly separation method described by Axmann et al. [29].Each prepared bedding sample was then divided into two equal portions, one of which was used immediately ("unstored") while the other was placed in a closed container stored in a controlled temperature (20˚C) room for 2 months prior to microcosm preparation ("prestored").Each bedding sample was regularly mixed manually during the storage period in order to limit heterogeneous development of anaerobic conditions.

Bedding microcosms experimental design
Four types of bedding material ("unstored straw", "pre-stored straw", "unstored straw + manure" and "prestored straw + manure") were used for preparing the bedding microcosms.The microcosms were prepared by placing 10 grams (wet weight) of bedding materials in a 720 ml whirl-pack1 bag, followed by incorporation of either 1.0 ml of freshly prepared S. enterica cocktail or 1.0 ml of freshly prepared E. coli O157:H7 cocktail.In uninoculated (control) microcosms, 0.85% saline was added in place of the bacterial cocktail.In total, 154 microcosms were inoculated with E. coli O157:H7, 154 microcosms were inoculated with S. enterica, and 174 microcosms were uninoculated, giving a total of 482 independent microcosms.All inoculated and uninoculated microcosm bags were left slightly open for sufficient aeration and incubated in a dark and temperature-controlled (20˚C) room.Microcosms were removed from the incubation room at the following intervals: 7, 13, 20, 35 or 70 days.At each sampling point, five to eight microcosms for each each inoculum and each bedding type were removed and immediately subjected to different analyzes (enumeration of E. coli O157:H7, enumeration of Salmonella spp., pH and moisture content) as listed in Table 1.In addition, at sampling points 0, 35 and 70 days, one to two microcosms for each inoculum and each bedding type were transferred into storage at -80˚C until DNA extraction.
The ambient air temperature (AT) and relative humidity (RH) of the incubation room were measured and recorded at 5 min interval during the incubation period using a data logger (tinytagTGP-4500; Gemini Data Loggers Ltd, Chichester, UK) positioned at approximately 0.5 m above the bags.Ambient AT and RH averaged throughout the incubation period were 21.5 ± 1.4˚C and 51.4 ± 8.1%, respectively.Moisture content of the bedding was determined using gravimetric analysis.Bedding pH was measured from the entire content of each bag suspended in 20 mL deionized water using a pHM210 MeterLabTM (Radiometer Analytical, Hach Lange NZ, Auckland, New Zealand).
Enumeration of E. coli O157:H7 and S. enterica E. coli O157:H7 and Salmonella spp.were enumerated from the bedding microcosms using a three-tubes three-dilutions MPN method, which is a method suitable for enumeration of viable organisms in low concentration and in presence of particulate matter [30].For enumeration of E. coli O157:H7, the entire content of the bedding microcosms were individually resuspended in 50 ml Modified Tryptic Soy Broth (mTSB; Fort Richard Laboratories) and mixed for 2 min in a Stomacher1 400 Circulator (Seward Limited, Worthing, United Kingdom).Serial dilutions of each suspended bedding were then prepared in mTSB, inoculated onto three 10 mL mTSB volume and incubated for 24h at 37˚C followed by 24 h at 42˚C.This primary enrichment was followed by selective plating on Cefixime tellurite sorbitol MacConkey agar (CT-SMAC; Fort Richard Laboratories) with plates incubated for 24 h at 42˚C.The presence of E. coli O157:H7 on CT-SMAC plates from the three most appropriate dilutions of the initial suspension was confirmed by RT-PCR RapidFinder STEC screen and RapidFinder O157 confirmation Assays (Applied Biosystems™ by Thermo Fisher Scientific, Life Technologies Corporation, Austin, USA).A similar protocol was used to enumerate S. enterica; bedding suspension and serial dilutions were prepared in Buffered peptone water (BPW; Fort Richard Laboratories), primary enrichment was in BPW for 18 h at 35˚C followed by Rappaport-Vassiliadis Salmonella enrichment medium (RVS; Fort Richard Laboratories) for 18 h at 42˚C, and selective plating was on Xylose Lysine Deoxycholate Agar (XLD; Fort Richard Laboratories) for 18 h at 35˚C.The presence of Salmonella spp. on XLD was confirmed by RT-PCR Salmonella confirmation Assay (Applied Biosystems™).The concentration of the bacterial target in the sample was determined by referencing the number of selective plates with confirmed bacterial growth to a three-tube MPN probability table [31].When the target was not detected (i. e., concentration below 4 (MPN).100g −1 ), a value of 0.01 (MPN).g - (fresh weight) bedding was assigned to the MPN count.The concentrations of E. coli O157:H7 and Salmonella spp.were expressed as Logarithms to avoid overestimation and were reported as Log 10 counts (MPN) g -1 (dry weight) bedding.

Microbial count analysis
Statistical analysis of E. coli O157:H7 and Salmonella spp.Log 10 counts were performed using two-way analysis of variance (ANOVA) in Genstat22 [32], with factors for pre-storage treatment and incubation time and the interaction term included.Fisher's protected least significant differences at the 5% level (LSD) were used to compare the means of unstored and 2 months pre-storage groups (geometric means on the back-transformed scale) at each sampling time.Data from straw with and without manure were analyzed independently.Residual diagnostic plots were checked for departures from the assumptions of normality and constant variance.

Bedding material data analysis
Mean moisture content and pH of the microcosm material was compared between unstored and pre-stored bedding over incubation time using a two-way analysis of variance (ANOVA), blocked by inocula (E. coli O157:H7, S. enterica or control), and fitted using Genstat22.The unstored and 2 months pre-storage means were compared at each incubation time using the LSD (5%).Data from straw with and without manure were analysed independently.Residual diagnostic plots were checked for departures from the assumptions of normality and constant variance.

DNA Extraction and sequencing
To ensure thorough mixing, the frozen straw retrieved from -80˚C storage was dried to constant weight at 105˚C for a maximum of one hour and homogenized in a Waring blender (model 8011ES, Waring commercial, Torrington, Connecticut, USA).Total DNA was extracted from 0.1 g of the very finely blended material using a QIAamp Fast Stool Mini kit (Qiagen Inc., Mississauga, Canada) according to the manufacturer instructions.Illumina 16S V3-V4 libraries were prepared from the extracted DNAs and were sequenced using an Illumina MiSeq instrument using chemistry version3 (Massey Genome Services, Palmerston North, New Zealand).

Bioinformatics methods
The processing of the amplicon reads followed a modified form of the pipeline described in [33].The reads produced by the sequencing instrument were paired using the program FLASH2 [34].Paired reads were then quality trimmed using Trimmomatic 0.38 [35].The trimmed reads were then reformatted as fasta, and the read headers were modified to include the sample name.All reads were compiled in a single file, and the Mothur program suit [36] was used to remove reads with homopolymers longer than 10 nt, and then to collapse the reads into unique representatives.The collapsed reads were clustered with the program Swarm [37].The clustered reads were filtered based on their abundance, keeping representatives that were a) present in one sample with a relative abundance >0.1%, b) present in >2% of the samples with a relative abundance >0.01%or c) present in 5% of the samples at any abundance level.The excluded OTUs (Operational Taxonomy Units) represented 6.5% of the reads, which suggested an acceptable quality of sequencing and data filtering.The selected OTUs were annotated using the Qiime program [38] with the Silva database v138 [39].The annotated tables were then used for downstream statistical analysis.The data was loaded into R version 4.1.1[40].Proportions were calculated for each superkingdom set by dividing the counts of each taxon by the total sample counts.Principal coordinates analysis (PCoA) was performed with the "APE" package version 5.5 [41] on Bray-Curtis distances [42] calculated using the "vegan" R package version 2.5-7 [43].Diversity estimates and ANOSIM [44] was also performed using the "vegan" R package with the default settings (including Bray-Curtis distance calculations).Permutation ANOVAs were performed using the lmPerm R package, version 2.1 [45] with 1 million permutations.
The numbers of inoculated S. enterica recovered from the microcosms are presented in Fig 1C and 1d (and S1 Table ).Immediately after inoculation, 7.1 Log 10 g -1 (95%CI: 6.0-8.2),7.0 Log 10 g -1 (95%CI: 5.9-8.1),7.2 Log 10 g -1 (95%CI: 6.6-7.8) and 6.4 Log 10 g -1 (95%CI: 5.8-7.0)were recovered from the unstored straw + manure, the pre-stored straw + manure, the unstored straw, and the pre-stored straw, respectively.In straw + manure, S. enterica decreased by 4.1 Log 10 g -1 (unstored) and 4.3 Log 10 g -1 (pre-stored) by incubation day 70, with no significant impact of pre-storage.In straw, S. enterica abundance remained for 35 days at levels comparable to or greater than the initial levels; by day 70, only a 1.3 (unstored) to 0.9 Log 10 (prestored) g -1 reduction of S. enterica was achieved.Naturally occurring Salmonella spp. was recovered from half of the control microcosms (12/21 straw + manure; 9/21 straw) but at concentrations consistently <5 MPN.g -1 The mean moisture content of the bedding material was stable over the incubation time in both prestored and unstored straw + manure microcosms, while it declined (p<0.001) in the straw microcosms (Table 2, S1 Table ).For the pre-stored straw + manure bedding, the mean pH values of bedding material were similar at day 0. By day 70, the mean pH of the pre-stored material was significantly (p<0.005)lower than that of the unstored material.For straw without manure, the mean pH increased over 70 days in both unstored straw and pre-stored material, with no difference in mean pH between stored and unstored straw at any time.Pre-storage of the bedding materials increased the number of bacterial taxa detected for each type of bedding; it significantly affected the richness of the microbial community (Chao1 (P = 0.01) and ACE (P = 0.01)), but not its eveness (Shannon (P = 0.65) and Simpson (P = 0.82) indexes) (Table 3).

Bacterial diversity and community profiles
Principal coordinates analyses (PCoA) based on Bray-Curtis indice revealed the bacterial communities in straw and straw + manure were significantly different (R = 0.46316, p<0.0001; perm = 1,000 analysis of variance using distance matrices) (Fig 2).In straw microcosms, a shift in the bacterial community was detected between days 0 and 35 in both prestored and unstored straw.Comparatively, the bacterial community in the straw + manure exhibited a more consistent diversity over time.The bacterial communities in straw + manure clustered by pre-storage at day 0, but not at day 35 and day 70.Neither the bacterial communities of the straw or that of the straw + manure were affected by the inoculation with E. coli O157:H7 or S. enterica.

Discussion
The study explored the effectiveness of straw animal beddings to inactivate E. coli O157:H7 and S. enterica introduced in the beddings after different storage time, and the possible role of the indigenous bacterial community.Results revealed that pre-storage of the bedding materials (for approx. 2 months prior to pathogen inoculation) was associated with a slower inactivation of E. coli O157:H7 (but not of S. enterica), and affected the composition but not the diversity of the indigenous bacterial community.
The two types of bedding materials (straw prior to use, or straw + manure) were sourced from a dairy farm at the end of the calving season.A cocktail of either four strains of E. coli O157:H7 or four strains of S. enterica was introduced in the bedding microcosms in order to include a possible strain-to-strain difference effect and determine the upper tolerance to inactivation; the high inoculum level simulated the worst-case pathogen loads possibly contaminating a bedding pile.Cattle, particularly calves and heifers, can excrete multiple genotypes of either E. coli O157:H7 or Salmonella spp., and at levels ranging from 10 2 to 10 7 CFU g −1 in feces [46][47][48][49].
The E. coli O157:H7 and S. enterica introduced into the experimental microcosms survived for the 10-week duration of the experiment, however E. coli O157:H7 numbers, and to a lesser extent Salmonella spp.numbers, declined over time.These findings were similar with those previously reported for abattoir wastes, bovine feces, or manure amended soils [23,50,51], where, in general terms, the population size of pathogenic bacteria was shown to progressively decline.Many studies that investigated persistence E. coli O157:H7 in different soils noted the importance of indigenous microbial diversity on the persistence of this pathogen.For example, Jiang et al. [51] and Baker et al. [52], who experimentally manipulated microbial diversity by sterilization and/or dilution-re-inoculation approaches, observed a greater survival of E. coli O157:H7 in autoclaved soils in comparison to unautoclaved soils.Survival time in autoclaved soils decreased with manure amendment [51,53].A more rapid decline of E. coli O157:H7 was reported in soils with an increasing history of solid manure addition [26].E. coli O157:H7 persistence was strongly and negatively correlated with indigenous bacterial richness [52,53].Similarly, the decline of S. enterica was found to be more rapid in soils amended with cover crops and compost that had higher bacteria diversity compared to the same soils treated with synthetic fertilizers and had a low bacterial diversity [54].Extrapolating the fate of zoonotic bacteria in soils to animal beddings needs to be done with caution, as there are many parameters that can affect microbial community -including C/N ratio, bulk density, water holding capacity, porosity or average particle size-that differ among soil, manure and straw-bedding [55].In the present study, the greatest decline of E. coli O157:H7 or S. enterica was observed when the bacterial community was highly diverse (e.g., in straw + manure), which is consistent with observations in soils.This indicates the negative impact of a diverse indigenous bacterial community on the persistence of zoonotic bacteria in animal bedding.Our study showed moreover that the inactivation of E. coli O157:H7 was slower when the bacteria were introduced into pre-stored bedding, confirming a previous observation in wastewater biosolids that the antagonistic effect of the indigenous bacterial community towards introduced food-borne pathogens declined with length of biosolids storage [56].Several mechanisms, including competition for resources, direct antagonism, or predation, have been proposed to explain the role of the resident bacterial community [54,57].
Habitats high in macronutrients or organic matter tend to contain more diverse bacterial communities and can positively affect suppression of foodborne pathogen through a greater use of resources [54].The survival of Shiga toxin-producing E. coli has been related to the number of carbon sources used by the resident microbial communities.This is because the higher the number of carbon sources used by the microbial communities, the lower the number that will be available for utilization by introduced E. coli strains [53].Ecosystems yielding a higher level of biodiversity are also in general less vulnerable to disturbances than ecosystems that have a lower diversity [58].Abundant interactions within a microbial community were shown to result in suppression of colonization by an invader E. coli O157:H7 [57,59].Our study found a greater initial shift in the bacterial community composition in straw compared to straw + manure, in which inactivation of the added pathogens appeared to be more effective.It is possible that the diverse bacterial community in the bedding containing manure was more stable to the perturbation associated with inoculation with the food-borne pathogens, and as a result more able to resist invasion.It is also possible that the E. coli O157:H7 or Salmonella were better able to survive when the bacterial community was "unstable" or less able to resist a perturbation, as was apparently the case for straw without manure.Further work is needed to better understand the biological mechanisms that result in inactivation of the introduced pathogens in perturbed ecosystems.
In the present study, the inactivation of both E. coli O157:H7 and S. enterica was concomitant to a reduction in Proteobacteria abundance.This finding differs from the negative correlation between survival of E. coli O157:H7 and Proteobacteria abundance established in soils and sandbased beddings [53,60].However, the influence of the different members within this phylum was shown to vary.For example, bacteria belonging to Beta-Proteobacteria order in particular have shown a suppressive effect on E. coli O157:H7, while other orders were thought to enhance survival [60].The efficiency of resource utilization by the "invader" was proposed to be the main driver, at least when considering species interactions on the basis of one resource at a time [61].In our study, close observation of the bacterial orders within the Proteobacteria showed dominance by Gamma-proteobacteria, such as Cellvibrionales, Burkholderiales and Xanthomonadales in straw+manure beddings, and by Xanthomonadales in straw.As these orders are known to degrade high-molecular weight or complex plant-based compounds, including cellulose, xylan, or starch [62], our study may indicate synergy between Proteobacteria and E. coli O157:H7 through use of available nutrient, rather than competition by antagonism.
The high abundance of Bacteroides observed when E. coli O157:H7 and Salmonella spp.are inactivated is in agreement with previous research in sand-based bedding and in soils that showed that the Bacteroides phylum as a whole exhibited a negative correlation with survival of E. coli O157:H7, with some specific species displaying a particularly suppressive effect [8,60].This is consistent with our finding that the relative abundance of the Sphingomonadales order was greater in prestored straw bedding where the inactivation of E. coli O157:H7 was slower, comparative to inactivation in unstored bedding.Bacteroidetes are known for their ability to initiate the degradation of complex [63], suggesting that the suppressive effect of Bacteriodes was possibly attributed to a more efficient use of complex lignocellulolytic resources.
Actinobacteria are present in a wide range of ecosystems and have a pivotal role in plant residues degradation, particularly in the less fertile environments [64,65].While the survival of zoonotic bacteria in soil has been found to be positively affected by Actinobacteria [60], some members of the Actinobacteria, including Nocardiopsis species, are also known to produce an array of bioactive compounds active against E. coli [66].In the present study, the comparison between stored and not stored beddings confirms that storage of straw + manure bedding might have affected nutrient availability but did not reveal a synergist or suppressive effect of Actinobacteria against Salmonella or E. coli O157:H7.
Compared to un-stored beddings, pre-stored bedding contained a greater number of bacterial taxa in relatively low abundance and that did not significantly affect the community structure.Examples include bacteria of the phyla Bdellovibrionota and Myxococcota, which have demonstrated an ability to enter into and lyse E. coli or Salmonella cells [67][68][69].However, the slower inactivation of E. coli O157:H7 in pre-stored beddings suggests that the increase in abundance of these bacterial phyla was unlikely to result in these phyla being present in number large enough to significantly affect zoonotic inactivation.Reasons for this could be a different relative location of target and low abundant predatory bacterial cells within the bedding material, or a specific physiological inability to parasitise E. coli O157:H7 or S. enterica in our experimental conditions.
As a general observation, the abundance of bacterial orders known to adapt to "dry" "salty" conditions increased over time on both beddings and was also greater in stored bedding compared to bedding that had not been stored.This suggests the possibility that salt levels increased during storage due to evaporation [54], which is in general agreement with the reduction of water content in stored beddings.In the current study, S. enterica tended to be inactivated less effectively than E. coli O157:H7 in both pre-stored bedding types, corroborating previous findings that Salmonella spp.better resists desiccation and other various environmental stresses than others zoonotic Enterobacteriaceae [70,71].The effect of water content alone on inactivation of Salmonella spp or E. coli O157:H7 in our study remains unclear.For instance, the longer survival of E. coli O157:H7observed on straw, which had a drier content than straw + manure, as well as the difference in inactivation between unstored and pre-stored straw + manure material despite their similar water content, suggests that the effect of water content on inactivation likely involves complex mechanisms.A similar unclear effect of water content on survival of E. coli O157:H7 was also reported in manure piles, with studies reporting a more frequent recovery of the bacteria from the middle and bottom of unaerated manure piles than from the top, and others reporting a longer survival time of pathogenic E. coli on the outside compared to the inside of the piles [21,72].Further studies considering the interaction effects between bedding-water content and nutrients availability or microflora population are required to better assess the effect of water content on bacterial inactivation and to develop recommandation for bedding management.
pH has been repeatedly shown to exert a critical role on the inactivation of E. coli O157:H7 and S. enterica in agricultural soils [24].In the current study, the inactivation of E. coli O157: H7 and S. enterica did not seem to be reflected by the pH changes in the neutral to alkaline gradient.We observed that higher pH values could produce longer survival time of E. coli O157: H7 strains, but also that, at high pH conditions, survival times were shorter when diversity of the bacterial community was high.It is likely that the effect of pH on survival time of E. coli O157:H7 in bedding was modulated by the bacterial community, as previously proposed for soil ecosystems [53].The increase in Bacteriodota and Actinobacteriota relative abundances in beddings with alkaline pH, which was expected given previous reports [73,74], suggests a particular link between these phyla, pH and E. coli O157:H7 or Salmonella spp.inactivation.Further investigation in animal beddings would be needed to determine the causal relationship between pH gradient, the presence and role of bacterial taxa and the inactivation of E. coli 157: H7 or Salmonella spp.
Care was taken at each step of our experiment to reduce the biological variability inherent to pastoral complex environments, and as a result, our experimental microcosms and pre-storage conditions did not fully reflect those in field or large-scale bedding heaps.In order to better guide effective bedding management practices or assess risks associated with the storage and recycling of animal beddings sourced from dairy farms, future studies should consider the inactivation of zoonotic bacteria when introduced in animal beddings at a low concentration, in association with organic matter, in different growth phase, or in different types of bedding substrates.Establishing a correlation between inactivation of zoonotic bacteria in pre-storage conditions representative of a range of farm practices and phylogenic data would also be critical to confirm key OTUs involved in lowering the levels of zoonotic bacteria.
In summary, this research contributed to the fundamental understanding of the fate of zoonotic bacteria introduced in animal beddings during storage and identified that bedding material storage conditions pre-and post use in animal facilities could be important to prevent the risk of zoonosis dissemination to the environment or to the dairy herds.For instance, slower inactivation of zoonotic bacteria in pre-stored beddings suggests that continuous addition of fresh manure or bedding on a stock pile would increase the risk of remaining zoonotic pathogens, and that a quarantine period during which no new manure or used bedding is added to the manure heaps should be observed before application on agricultural land.Sporadic detection of the two pathogens at low concentration in the straw material prior to use in the animal facility suggests a point contamination, such as wildlife excreta.Birds and rodents can contribute to the cycling of food-borne bacteria on farm [75], and implementation of biosecurity measures targetting wildlife around the storage areas of bedding material prior to use in animal facilities should be encouraged to control the risks of recontaminating the animals or introducing new strains in the herds.In terms of maturation practices, our findings suggest that storage of used animal beddings under conditions promoting a diverse and stable bacterial community might improve zoonosis inactivation.Overall, development of guidelines on bedding storage management pre and post use in animal facilities and impact on pathogens inactivation are recommended.

Fig 1 .
Fig 1. Inactivation of E. coli O157:H7 and S. enterica in unstored (filled diamond) and pre-stored (hollow circle) straw +manure bedding (a, c) and straw (b, d).Each MPN counts were obtained from triplicate bags.Bars are 95% confidence intervals for the geometric mean counts.* indicates significant difference at p = 0.05 between unstored and pre-stored geometric mean counts within an incubation time.https://doi.org/10.1371/journal.pone.0295843.g001 Bacterial community profiling was carried out on each type of bedding after inoculation of the bacterial cocktail (day 0), and on days 35 and 70.A total of 1,0365,141 read pairs of 300 nt were obtained from the high-throughput sequencing of the 68 samples.The filtered reads were clustered into 718 species-level OTUs.Bacterial communities were reasonably characterized with the sampling effort for all bedding types as the rarefaction curves of the filtered OTUs approached horizontal (S1 Fig).More OTUs were represented in the straw + manure bedding in comparison to the straw bedding (p < 0.01) (S1 Fig).

Fig 2 .
Fig 2. Bray-Curtis principal coordinate analysis (PCoA) illustrating the changes in bacterial community betadiversity in the four beddings types over incubation time.Each symbol represents a sample, with distance between samples calculated using Bray-Curtis similarity measures.Dashed ellipses indicate 95% confidence intervals of the ordinations for named clusters.https://doi.org/10.1371/journal.pone.0295843.g002

Fig 3 .
Fig 3. Change of relative abundance of major bacterial phyla across bedding types (a: straw; b: straw + manure) with and without pre-storage treatment and over incubation time.Only the phyla with abundance above 1.0% were plotted.Bars are standard error of mean (n = 6).https://doi.org/10.1371/journal.pone.0295843.g003

Table 1 . Bedding microcosms experimental design with the number of microcosms used for each analyses. Bedding type Inoculum a Sampling points (days of incubation) No of microcosms analyzed at each sampling point material pre-storage treatment E. coli O157 counts Salmonella counts pH moisture Bacterial community
b not done at day 20 c not done at days 13 and 35 d not done at days 7 and 20 e one microcosm analyzed at day 35 and at day 70 https://doi.org/10.1371/journal.pone.0295843.t001